gbpr principles of biotechnology.pdf

8/17/2019 GBPR principles of biotechnology.pdf

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DEPARTMENT OF GENETICS AND PLANT BREEDING

1. Course No. : GBPR 212

2. Course Title : Principles of Plant Biotechnology

3. Credit Hours : 3 (2+1)

4. General Objective : To impart knowledge to the students on the

various techniques of plant tissue culture,

principles of plant biotechnology and their role

in crop improvement

5. Specific Objectives

Theory

By the end of the course, the students will be able to

i. understand the various techniques of plant tissue culture

ii. know about the fundamentals of genetic engineering

iii. study about molecular markers, Quantitative Trait Loci (QTL) mapping and MarkerAssisted Selection

Theory Lecture Outlines

1. Biotechnology – definitions – major concepts and importance – international

organizations involved in biotec hnology – biotechnology in India

2. History of plant tissue culture and plant genetic engineering – terminology used in plant

tissue culture

3. Plant cell and tissue culture – steps in general tissue culture techniques – merits and

limitations – applications of plant tissue culture in crop improvement

4. Different techniques used for sterilization in plant tissue culture, growth room chambersand instruments

5. Nutritional requirements of tissue culture – preparation and composition of Murashige

and Skoog (MS) medium

6. Types of media – solid and liquid media – advantages and limitations – types of

cultures – callus and suspension cultures

7. Totipotency and morphogenesis – growth and differentiation in cultures –

8. Micropropagation – meristem culture – procedure – various approaches for shoot

multiplication

9. Micropropagation – applications – problems – advantages and limitations10. Somaclonal variation – types – origin – applications – advantages – limitations –

achievements

11. Anther / pollen culture – brief procedure – factors affecting androgenesis

12. Anther / pollen culture – applications of haploids in crop improvement – limitations –

achievements

13. Embryo culture – purpose – methods of embryo culture – procedure – applications –


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achievements – ovule culture – ovary culture

14. Endosperm culture – purpose – procedure – applications

15. Somatic embryogenesis – stages of somatic embryo development – general procedure –

factors affecting somatic embryogenesis – applications – limitations

16. Artificial seed / synthetic seed production – desiccated systems and hydrated systems of

synthetic seed production – advantages and limitations

17. In vitro pollination and fertilization – factors affecting in vitro pollination – applications

18. Protoplast culture – methods of protoplast isolation – culture of protoplasts – somatic

hybridization – procedure – isolation, culture, fusion of protoplasts, selection and

culture of somatic hybrid cells and regeneration of hybrid plants

19. Somatic hybridization – products of somatic hybridization – symmetric hybrids,

asymmetric hybrids and cybrids – advantages and limitations of somatic hybridization

20. Genetic engineering – definition – general approach for genetic engineering in plants –

risks of genetic engineering

21. Method of cloning DNA in bacteria – steps involved in gene cloning – components of

gene cloning and their functions

22. Restriction enzymes – types – nomenclature – cleavage patterns and applications

23. Vectors for gene transfer – properties of a good vector – cloning and expression vectors

– plasmids, cosmids, bacteriophages, phagemids, Yeast Artificial Chromosome (YAC),

Bacterial Artificial Chromosome (BAC) and Shuttle vectors

24. Isolation of DNA fragments – genomic libraries and Complementary Deoxy

Ribonucleic Acid (cDNA) libraries – detection of a gene with in a library – colony

hybridization – procedure and applications of blotting techniques – southern blotting (in

detail), northern blotting and western blotting – comparison of blotting techniques – probes – definition and applications

25. Polymerase Chain Reaction (PCR) – procedure and applications – comparison of PCR

and gene cloning

26. Molecular markers – definition – brief description of different types of DNA based

markers – Restriction Fragment Length Polymorphism (RFLP), Amplified Fragment

Length Polymorphism (AFLP), Randomly Amplified Polymorphic DNA (RAPD) and

Simple Sequence Repeats (SSR) markers – importance, procedure and applications

27. DNA fingerprinting – applications; Quantitative Trait Loci (QTL) mapping – MarkerAssisted Selection (MAS) and its applications in crop improvement

28. Methods of gene transfer – indirect method of gene transfer – Agrobacterium mediatedgene transfer method

29. Methods of gene transfer – direct methods of transformation – particle bombardment /

gene gun method, chemical method, electroporation, lipofection, microinjection,

macroinjection, pollen transformation, delivery via growing pollen tubes, laser induced,

fiber mediated transformation etc.

30. Transgenic plants – applications in crop improvement – limitations


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31. Genetic engineering for insect resistance – introduction of resistance genes from micro-

organisms (Cry gene from Bacillus thuringiensis – Bt cotton) and higher plants

(protease inhibitors, amylase inhibitors and lectins); Genetic engineering for herbicide

resistance – detoxification and target modification; Genetic engineering for resistance

to diseases caused by virus (coat-protein-mediated resistance, satellite RNAs mediated

resistance and Antisense RNA mediated resistance), fungi (antifungal protein mediated

resistance and antifungal compound mediated resistance) and bacteria

32. Genetic engineering for male sterility – barnase / barstar system; Genetic engineering

for quality modifications and novel features – golden rice slow fruit softening tomato

(FlavrSavr tomato)

References

Bilgrami, K.S. and Pandey, A.K. 1992. Introduction to Biotechnology. CBS Pub., New

Delhi.

Chahal, G.S. and Gosal, S.S. 2002. Principles and Procedures of Plant Breeding –

Biotechnological and Conventional Approaches. Narosa Publishing House, NewDelhi.

Chawla, H.S. 2005. Introduction to Plant Biotechnology. Oxford and IBH Publishing Co.,

New Delhi.

Gupta, P.K. 1994 Elements of Biotechnology. Rastogi and Co., Educational Publishers,Meerut.

Jha, T.B. and Ghosh, B. 2005. Plant Tissue Culture. University Press, Hyderabad.

Razdan, M. K. 2002. Introduction to Plant Tissue Culture. Oxford and IBH Publishing

Co., New Delhi.

Singh, B.D. 2006. Plant Biotechnology. Kalyani Publishers, Ludhiana.


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Lecture. No. 1

Biotechnology – Definitions – Major concepts and importance –International organizations involved in biotechnology –

Biotechnology in IndiaIntroduction

The term Biotechnology was coined by karl Ereky a Hungarian engineer in 1919. This

term is derived from a fusion of Biology and Technology

Biotechnology is not a pure science but an integrated affect of these two areas, the root

of which lies in biological sciences

It is truly multidisciplinary in nature and it encompasses several disciplines of basic

sciences and engineering

The science disciplines from which biotechnology draws heavily are Microbiology,

Biochemistry, Chemistry, Genetics, Molecular biology, Immunology and Physiology

On engineering side it leans heavily on processes chemical and biochemical

engineering since large multiplication of microorganisms and cells their down stream

processing etc. are based on them.

It is a fast growing science and it has been defined in different ways by different group

of workers.

Definition

Biotechnology is the application of scientific and engineering principles to the

processing of materials by biological agents to provide goods and services. This was given by

OECD – the organization for economic cooperation and development in 1981.

Although the term was recent origin the discipline itself is very old. Man began

employing microorganisms as early as in 5000 B.C for making wine vinegar curd etc.

All these processes which are based on the natural capabilities of microorganisms are

commonly considered as old biotechnology. The development of recombinant technology

allowed to modify microorganisms and other organisms to create in them highly valuable,

novel and naturally non-existing capabilities. Eg:- The human gene producing Insulin has

been transferred and expressed in bacterium like E.coli and it is being used in management ofDiabetes. Crop varieties and animal breeds with entirely new and highly useful traits are being

created with the help of recombinant DNA technology

These and many other similar examples constitutes modern the new Biotechnology in

India.

In 1982 Government of India set up an official agency National Biotechnology Board

(NBTB) which started functioning under the Department of Science and Technology (DST)


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In 1986 NBTB was replaced by a full fieldged department, the Department of

Biotechnology (DBT) in the ministry of sciences. Technology for planning, promotion and

coordination of various biotechnological programmes.

More over on the proposal of United Nations Organization (UNO) the International

Center of Genetic Engineering and Biotechnology (ICGEB) was established to help the

developing countries. ICGEB has its two centers one in New Delhi and the other in Trieste

(Italy)

The New Delhi center of ICGEB is functioning proper way since in 1988

The other central organizations for Biotechnology research in India are

IARI : Indian Agricultural Research Institute, New Delhi

JNU : Jawaharlal Nehru University, New Delhi

IVRI : Indian Veterinary Research Institute, Izatnagar

CFTRI : Central Food Technology Research Institute, Mysore NDRI : National Dairy Research Institute - Karnal - Haryana

MRC : Malaria Research Center – New Delhi

RRL : Regional Research Laboratory – Jammu

CDRI : Central Drug Research Institute – Lucknow

CIMAP : Central Institute of Medicine and Aromatic plants - Lucknow and Hyderabad

IIT : Indian Institute of Technology – Kanpur, New Delhi

IISC : Indian Institute of sciences – Bangalore

IMTECH : Institute of Microbial Technology – Chendiger

NIM/NII : National Institute of Immunology – New Delhi

NCL : National Chemical Laboratory – Pune

CCMB : Center for Cellular and Molecular Biology – Hyderabad

CDFD : Center for DNA Finger Printing and Diagnostics – Hyderabad

CPMB : Center for Plant Molecular Biology – 7’ centers

BARC : Baba Atomic Research Center – Mumbai

Other international research centers programmes involved in biotechnology

UNEP : United Nations Environment Programme

ICRO : International Cell Research organization

IIB : International Institute of Biotechnology – Conterbury kent in UK


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Abbreviations used in Biotechnology

PAGE : Poly Acrylamide Gel Electrophoresis

RFLP : Restriction Fragment Length polymorphism

RAPD : Randomly Amplified polymorphic DNA

cDNA : Complementary DNA

mt DNA : Mitochondrial DNA

PCR : Polymerase Chain Reaction

HPLC : Higrowth hormone Performance Liquid Chromatography

PEG : Poly Ethylene Glycol

HFCS : Higrowth hormone Fructose Corn Syrup

HEPA : Higrowth hormone Efficiency Particulate Air

GMO : Genetically Modified OrganismsGm foods / Gm crops.

MAS : Marker Assisted Aided Selection

ELISA : Enzyme Linked Immuno Sorbent Assay

NAA : Napthelene Acetic Acid

IAA : Indole – 3 - Acetic acid

IBA : Indole – 3 – Butyric acid

BAP : Benzyl Amino Purine

BA : Benzyl Adenine

Ti plasmid : Tumer inducing

HGH : Human Growth Harmone

SSRs : Simple Sequence Repeats

QTL : Quantitative Trait loci

VNTRS : Variable Number of Tandem Repeats.

GEAC : Genetic Engineering Approval Committee

GEM : Genetically Engineered Micro Organism

CMV : Cauliflower Mosaic Virus

TMV : Tobacco Mosaic Virus

STS : Sequence Tagged Sites

tDNA : Transferred DNA

EDTA : Ethylene Diamine Tetra Acetic acid.

Pg : Picograms

ppm : Parts Per Million

MOET : Multiple Ovule and Embryo transfer


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Lecture No. 2*

History of plant tissue culture and genetic engineering –

Terminology used in plant Tissue Culture

The term ‘plant tissue culture culture’ broadly refers to the in vitro cultivation of

plants, seeds, plant parts on nutrient media under aseptic conditions.

During the 1800s, the cell theory (Schleiden and Schwann) which states that the cell is

the basic structural unit of all living organisms, was very quick to gain acceptance. However,

the second portion of the cell theory states that these structural units are distinct and

potentially totipotent physiological and developmental units, failed to gain universal

acceptance.

In 1902, Gottlieb Haberlandt, a German plant physiologist, attempted to cultivate plant

tissue culture cell in vitro. He is regarded as the father of plant tissue culture. Totipotency is

the ability of plant cell to perform all functions of development, which are characterstic of

zygote i.e its ability to develop into a complete plant. In 1902, Haberlandt attempted culture

of isolated single palisade cells from leaves in knop’s salt solution enriched with sucrose. The

cells remaine d alive for up to one month, increased in size, accumulated starch but failed to

devide Demonstration of totipotency led to the development of techniques for cultivation of

plant cells under defined conditions.

The first embryo culture, although crude, was done by Hanning in 1904.

In 1925 Laibach recovered hybrid progeny from an inter specific cross in Linum.

In 1964 Maheshwari and Guha were first produced haploid plants from pollen grains, by

culturing anthers of Datura.

In 1960, cocking isolated protoplast for culturing.

In 1972 Carlson et al produced first somatic hybrid plants by fusing the protoplasts of

N. glauca x N. langsdorfli


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History of Biotechnology

Year Name of the

Scientist(s)

Important contribution

1902 Haberlandt First attempt of plant tissue culture (Father of Plant

Tissue culture)

1904 Hannig First attempt to culture embryo of selected crucifers

1922 Knudson Asymbiotic germination of orchid seeds in vitro

1922 Robbins In vitro culture of root tips

1925 Laibach Use of embryo culture technique in interspecific

crosses of linseed ( linum)

1934 Gautheret In vitro culture of the cambial tissue of a few trees and

shrubs, although failed to sustain cell division.1934 White Successful culture of tomato roots

1939 Gautheret, Nobecourt

and White

Successful establishment of continuously growing

callus cultures

1940 Gautheret In vitro culture of cambial tissues of Ulmus to study

adventitious shoot formation

1941 Van Overbeek Use of coconut milk containing a cell division factor

for the first time to culture Datura embryos

1941 Braun In vitro culture of crown gal tissues

1944 Skoog In vitro adventitious shoot formation in tobacco

1946 Ball Raising of whole plants of Lupinus and Tropaeolum by

shoot tip culture

1950 Ball Regeneration of organs from callus tissue of Sequoia

sempervirens

1952 Morel and Martin Use of meristem culture to obtain virus-free Dahlias

1952 Morel and Martin First application of micrografting

1953 Tulecke Production of haploid callus of the gymnosperm

Ginkgo biloba from pollen

1954 Muir et al First plant regenerated from a single cell

1955 Miller et al Discovery of kinetin, a cell division hormone

1956 A, Kornberg et al In vitro synthesis of DNA


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1957 Skoog and Miller Discovery of the regulation of organ formation by

changing the ratio of auxin : cytokinin

1958 Maheshwari and

Rangaswamy

Regeneration of somatic embryos in vitro from the

nucellus of Citrus ovules

1959 Reinert and Steward Regeneration of embryos from callus clumps and cell

suspensions of carrot ( Daucus carota )

1959 Gautheret Publication of first handbook on “Plant Tissue

Culture”

1960 Kanta First successful test tube fertilization in papaver

rhoeas

1960 E. Cocking Enzymatic degradation of cell walls to obtain large

number of protoplasts

1960 Bergmann Filtration of cell suspensions and isolation of single

cells by plating

1962 Murashiqe and Skoog Development of Murashige and Skoog nutrition

medium

1964 Guha and Maheshwari Production of first haploid plants from pollen grains of

Datura (Anther culture)

1968 H.G. Khorana Awarded Nobel prize for deciphering of genetic code

H.G. Khorana et al. Deduced the structure of a gene for yeast alanyl tRNA

1968 Meselson and Yuan Coined the term “Restriction endonuclease” to describe

a class of enzymes involved in cleaving DNA

1970 Carlson Selection of biochemical mutants in vitro by the use of

tissue culture derived variation

1970 Power et al. First achievement of protoplast fusion

1970 H. Temin and D.

Baltimore

Discovered the presence of reverse transcriptase (a

RNA directed DNA polymerase which has the ability

to synthesize cDNA using mRNA as a template1970 Smith Discovery of first restriction endonuclease from

Haemophillus influenzae Rd. It was later purified and

named Hind 11


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1971 Nathans Preparation of first restriction map using Hind II

enzyme to cut circular DNA or SV 40 into 11 specific

fragments

1971 Takebe et al, Regeneration of first plants from protoplasts

1972 Carlson et al, First report of interspecific hybridization through

protoplast fusion in two species of Nicotiana

1972 Berg et al, First recombinant DNA molecule produced using

restriction enzymes

1974 Reinhard Biotransformation in plant tissue cultures

1974 Zaenen et al. ; Larebeke

et al.

Discovered the fact that the Ti plasmid was the tumor

inducing principle of Agrobacterium

1976 Seibert Shoot initiation from cryo-preserved shoot apices of

carnation

1976 Power et al. Inter-specific hybridization by protoplast fusion or

Petunia hydrida and P. parodii

1977 Maxam and Gilbert A method of gene sequencing based on degradation of

DNA chain

1977 Sharp and Roberts Discovery of split genes

1978 Melchers et al. Somatic hybridization of tomato and potato resulting in

pomato

1979 Marton et al. Co-cultivation procedure developed for transformation

of plant protoplasts with Agrobacterium

1980 Alfermann et al Use of immobilized whole cells for biotransformation

of digitoxin into digoxin

1980 Eli Lilly and Co. Commercial production of human insulin through

genetic engineering in bacterial cells

1981 Larkin and Scowcroft Introduction of the term somaclonal variation

1982 Krens et al. Incorporation of naked DNA by protoplast resulting inthe transformation with isolated DNA

1982 Zimmermann Fusion of protoplasts using electric stimuli

1983 Kary B. Mullis Conceived the idea of Polymerase chain reaction

(PCR), a chemical DNA amplification process


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1983 Pelletier et al. Intergeneric cytoplasmic hybridization in Raddish and

Grape

1984 De Block et al.; Horsch

et al.

Transformation of tobacco with Agrobacterium;

transgenic plants developed

1984 Alec Jeffreys Development of the genetic fingerprinting technique

for identifying individuals by analyzing polymorphism

at DNA sequence level

1986 Powell-Abel et al. TMV virus-resistant tobacco and tomato transgenic

plants developed using cDNA of coat protein gene of

TMV

1987 Sanford et al.; Klein et

al.

Development of biolistic gene transfer method for

plant transformation

1987 Barton et al. Isolation of Bt gene for bacterium

( Bacillus thuringiensis)

1990 Formal launch of the Human Genome Project

1990 Williams et al.; Welsh

and McClelland

Development of the Random Amplified Polymorphic

DNA (RAPD) technique

1991 Fodor Development of DNA microarray system using light

directed chemical synthesis system

1995 Fleischmann et al. Reporting by the institute for Genomic Research of the

complete DNA sequence of Haemophilus influenzae

1995 Vos et al. Development of DNA fingerprinting by Amplified

Fragment Length Polymorphism (AFLP) technique

1997 Blattner et al. Sequencing of E. coli genome

1998 C. elegans sequencing

consortium

Sequencing of the genome of a multicullular organism

(Caenorhabditis elegans)

2001 Human Genome Project

Consortium and Venteret al.

Sequencing of human genome successfully completed


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Terminology used in plant Tissue culture

The term ‘Plant Tissue Culture’ broadly refers to the in vitro cultivation of plants,

seeds, plant parts etc. on nutrient media under aseptic conditions.

Haberlandt (1854-1945) attempted to cultivate plant tissue culture cells in vitro. He is

regarded as the father of plant tissue culture.

Plant tissue culture:- Common term used to cover all types of aseptic plant cultures

Culture:- Growing of cells tissues plant organs (or) whole plants in nutrient medium under

aseptic conditions. Depending upon the explant source it can be named as follows :

Anther : anther culture

Pollen : pollen culture

Embryo : Embryo culture

Cell : Cell culture

Protoplast : Protoplast cultureCallus : callus culture

Organ culture : Culture of isolated plant organs such as root tips, shoot tips, leaf

primordial, immature parts of flower etc.

Cell culture : culturing of single cell (or) a small group of similar cells.

Aseptic culture : Arising of culture from a tissue (or) an organ after elimination of

bacterium, fungi and micro organism

Suspension culture : Culturing of cells (or) cell aggregates in liquid medium

Batch culture : Cell suspension grown in fixed volume of liquid Medium

Continuous culture : A suspension culture continuously supplied with nutrients by

continuous flow of fresh medium. The volume of culture medium is

normally constant

Nutrient medium : A solid (or) liquid combination of nutrients and water usually

including several salts, carbohydrates in the form of sugar and

vitamins such a medium is called basal medium. The basal medium

may be supplemented with growth harmone occasionally with other

defined and undefined substances.

Auxins : A class of growth hormone which cause cell elongation, apical

dominance, root initiation Eg : NAA, IAA, 2.4-D

Cytokinens : A class of growth hormone which cause cell division, shoot

differentiation, breaking of apical dominance etc. kinetin, zeatin etc.


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Explant : A piece of tissue used to initiate tissue culture or removing shoots

from callus separating individual shoots from proliferating mass of

shoots.

Callus : A tissue arising from disorganized proliferation of cells either in

culture (or) in nature

Sub culture : Aseptic transfer of a part of a culture to a fresh medium

Passage time : The time interval between two successive sub cultures

Clone : A population of cells derived from single cell by mitotic division (or) A

propagation of plants derived from a single individual plant througrowth

hormone vegetative propagation / genetically identical.

Clonal propagation : Asexual multiplication starting from single individual

Micro propagation : Production of minia ture planting material (somatic embryos (or)

plantlets) in large number by vegetative multiplication through growthhormone regeneration

Totipotency : The ability inherent property of a cell (or) tissue to give rise to whole plant

irrespective of their ploidy level and the form of specialization

Meristem : A group of actively dividing cells from which permanent tissue systems

such as root, shoot, leaf, flower etc are derived

Meristemoid : A gp of meristematic cells with in a callus with a potential to form primordial

Embryoid / Somatic embryos : Non zygotic embryo’s formed in culture.

Organogenesis : Type of morphogenesis which results in the formation of organs and / or

origin of shoots roots. The floral organs from tissue culture (or)

suspension culture

Regeneration

In tissue culture it is used for development of new organs (or) plantlets from a tissue,

callus culture (or) from a bud.

Embryogenesis

The process of embryo initiation origin of plantlet in a developmental pattern that

closely resembles the normal embryo development from fertilized egg or ovum.

I n vivo : a latin word literally means in living applied to any process occur in a whole

organism under field condition where there is no control over the environmental

conditions

I n vi tro : A latin word literally means in glass / living in test tube applied to any process

carried out in sterile culture under controlled condition in the laboratory


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Amplification : Creation of many copies of a segment of DNA by PCR / Duplication of

genes within a chromosomal segment.

Parasexual hybridization : Hybridization by non-sexual methods. Eg:- protoplast fusion

Cybrid : Plant (or) a cell which is a cytoplasmic hybrid produced by fusion of protoplast

cytoplast

Protoplast: A single cell with their cell walls stripped off a cell without a cell wall

Cytoplast: Protoplast – nucleous enucleated protoplast

Heterokaryon: A cell in which two or more nuclei of unlike genetic make up are present

Homokaryon: A cell with two or more nuclei of similar genetic make up

Synkaryon: Hybrid cell produced by fusion of nuclei in heterokaryon

Hetroplast : Cell containing foreign organells

Genetic Engineering

Manipulation of genetic architecture of an organism of DNA level (or) molecular level

rDNA : Recombinant:- The DNA which contains gene from different sources and can

combine with DNA of any organism

Transgenic plants: Plants which contain foreign DNA

Lecture No. 3*

Plant cell and tissue culture

Steps in general tissue culture techniques – merits and limitations –

Application of plant tissue culture in crop improvement

Plant tissue culture is the aseptic method of growing cells and organs such as

meristems, leaves, roots etc either in solid or liquid medium under controlled condition. In

this technique small pieces of viable tissues called ex-plant are isolated from parent plants and

grown in a defined nutritional medium and maintained in controlled environment for

prolonged period under aseptic condition.

The general technique of plant tissue culture involve four main stages. They are

Initiation of cultureMultiplication (or) sub culture

Development and differentiation

Hardening

1. Intiation of culture


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The most important factor in tissue culture technique is the maintenance of aseptic

condition. For this purpose the culture medium generally, a GR-free medium is used

Immediately after preparation the culture vessel has to be plugged and autoclaved at 121OC

15 psi (pounds per sq. inch) for an about 15-20min. The plant material has to be surface

sterilized with a suitable sterilent. The transfer area should also maintained free of micro

organisms. Strict precautions are to be taken to prevent the entry of micro organisms.

The plug of a culture vessel is removed carefully to transfer plant material to the nutrient

medium during sub culturing. After inoculation the cultures are incubated in culture room

under controlled condition at 25 + 12OC temperature and 1000 lux light intensity generated by

florescent tube and at a constant photoperiod regulated by automatic timers.

2. Multiplication / Subculture

After 2-3 weeks the explants show visible growth by forming either callus (or)

differentiated organs like shoots, roots (or) complete plantlets, depending upon the

composition of the medium. Periodically sub-culturing of callus (or) organs (or) plantlets to

the fresh medium is done to multiply the callus (or) organs (or) to obtain large number of

plantlets from the callus.


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3. Development and Diffentiation / organogenesis

The concentration of phytoharmones in the medium are altered to induce

differentiation in callus. A high cytokinins to auxin ratio induces shoot formation

(caulogenesis) (basal medium + low cytokinins / GA3 medium is used before they can be

rooted. Higher concentration (>2 mg/l BAP) of cytokinins induce adventitious shoot buds and

retard shoot growth. Very high auxins to cytokinin ratio induces root formation

(Rhizogenesis). The development of organ structures like shoot, roots etc. from the cultured

cells (or) tissues is known as organogenesis. Alternatively media composition can also be

altered to induce the development of somatic embryos and the process is known as somatic

embryogenesis. Further, an entire plantlet can be induced to grow on culture media by

manipulating the phytoharmone balance correctly and the process is called Regeneration. The

regeneration may be either direct or callus mediated. The in vitro induced shoots must be

transferred to the culture media that supports root induction.

Steps in plant tissue culture technique

Selection of plant

Isolation of explant

Sterilization of explant

Inoculation of explant

Incubation

Initiation of callus

Sub culturing

Regenaration

Hardening

Transfer of plantlets to Green house or open field


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4) Hardening:

The in vitro cultured rooted plants are first subjected to acclimatization before

transferring to the field. The gradual acclimatization of in vitro grown plant to in vivo

conditions is called hardening. The plantlet is taken out from the rooting medium and is

washed thoroughly to remove entire agar from the surface of plantlet as agar may attract

microbes to grow and destroy the plantlets. The plantlet is now kept in a low minimal salt

medium for 24-48hrs and transferred to a pot that contains autoclaved sterilized mixture of

clay soil, coarse sand and leaf moulds in 1 : 1 : 1 ratio proportion. The pot containing plantlet

is covered generally with the transparent polythene cover having holes for aeration to

maintain the humidity. The plantlets are maintained for about 15-30 days in this condition.

The plantlets are then transferred to the soil and are ready for transfer either to the green

house or main field.

Applications of plant tissue culture in crop improvement1. Micro propagation helps in mass multiplication of plants which are diffic ult to

propagate through conventional methods.

2. Some perennial crop plants like ornamental and fruit crops can not be propagated

through seeds. The vegetative propagation like grafting, budding are tedious and time

consuming. In such crops micro propagation helps in rapid multiplication.

3. Rapid multiplication of rare and elite genotypes such as Aromatic and Medicinal plants.

Isolation of in vitro mutants for a large number of desirable character Eg:- Isolation of

biochemical mutants and mutants resistant to biotic (pest and disease) abiotic (salt and

drought, cold, herbicide etc) stresses through the use of somaclonal variation

4. Screening of large number of cells in small space.

5. Cross pollinated crops like cordamum, Eucalyptus, coconut, oil palm do not give true to

type plants, when multiplied through seed. Development of genetically uniform plants in

cross pollinated crops is possible through tissue culture

6. In case of certain horticultural crops orchids etc seed will not germinate under natural

conditions, such seed can be made to germinate in vitro by providing suitable

environment.

7. Induction of flowering in some trees that do not flower or delay in flowering.

Eg:- Bamboo flowers only once in its life time of 50 years

8. Virus free plants can be produced through meristem culture


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9. Large amount of germplasm can be stored within a small space and lesser cost for

prolonged periods under in vitro condition at low temperature. The preservation of cells

tissues, organs in liquid Nitrogen at – 196OC is called cryopreservation

10. Production of secondary metabolites. Eg:- Caffine from coffea arabica, Nicotine from

Nicotiana rustica.

11. Plant tissue culture can also be used for studying the biochemical pathways and gene

regulation.

12. Anther and pollen culture can be used for production of halploids and by doubling the

chromosome number of haploids using cholchicine homogygous diploids can be

produced. They are called dihaploids.

13. In case of certain fruit crops and vegetative propagated plants where seed is not of much

economic important, triploids can be produced through endosperm culture.

14.

Inter specific and inter generic hybrids can be produced through embryo rescuetechnique which is not possible through conventional method. In such crosses in vitro

fertilization helps to overcome pre-fertilization barrier while the embryo rescue

technique helps to over come post fertilization barrier.

15. Somatic hybrids and cybrids can be produced through protoplast fusion (or) somatic

hybridization

16. Ovary culture is helpful to know the physiology of fruit development.

17. Development of transgenic plants.

Advantages of tissue culture

Rapid multiplication within a limited space

It is not time bound and not season bound

Free from pests and diseases

Limitations (or) Disadvantages

Laborious, costly, special risk is required.

Lecture No. 4*

Different types of techniques used for sterilization in plant tissue culture,growth room chambers and instruments

The media used for plant tissue culture contain sugar as a carbon source there by

attracting a variety of micro organisms including bacteria and fungi. These organisms grow

much faster than the cultured tissues and produce metabolic substances which are toxic to

plant tissues. There are a number of sources through which the media may get contaminated


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which include culture vessels, instruments, media, explant, transfer area and culture room.

There fore sterilization is absolutely essential to provide and maintain a completely aseptic

environment during in vitro cultivation of plant cells (or) organs.

Sterilization is a procedure used for elimination of micro organisms and maintaining

aseptic (or) sterile conditions for successful culture of plant tissues (or) organs.

The different techniques used for sterilization of plant tissue culture growth room

chambers and instruments are

1. Dry sterilization

2. Wet heat / autoclaving / steam sterilization

3. Ultra filtration (or) Filter sterilization

4. Ultra violet sterilization

5. Flame sterilization

6.

Surface sterilization (or) chemical sterilization7. Wiping with 70% alcohol

1. Dry heat sterilization : Empty glass ware (culture vessels, petriplates etc) certain

plastic ware (Teflon, FFp), Metallic instruments (scalpels, foreceps, needles etc)

aluminium foils, paper products can be sterilized by exposure to hot dry air at 160O

-

180OC for 2-4hr in hot air oven. All items should be properly sealed before sterilization.

2. Wet heat sterilization (or) autoclaving steam sterilization : It is a method of

sterilization with water vapour under high pressure to kill all microbes by exposing to

the super heated steam of an autoclave. Normally the tissue culture media in glass

containers sealed with cotton plugs (or) Aluminium, foils, plastic caps are autoclaved

with a pressure of 15psi at 121OC for 15-20 minutes. From the time the medium reaches

the required temperature some types of plastic glassware can also be repeatedly

sterilized by autoclaving (Good sterilization relies on time, pressure, temperature and

volume of the object to be sterilized).

The advantages of an autoclave are speed, simplicit y and distruction of viruses, while

disadvantages are change in pH by 0.3 – 0.5 units.

3.

Ultra filteration / Filter sterilization : Vitamins, amino acids, plant extracts,

harmones, growth Regulators are thermolabile and get destroyed during autoclaving.

Such chemicals are filter sterilised by passing through a bacterial proof membrane filter

under positive pressure. A millipore (or) seitiz filter with a pore size of not more than

O,2µ is generally used in filter sterilization. This procedure has to be carried out only in


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aseptic working space created by laminar air flow cabinet. Filter sterilised thermolabile

compounds are added to an autoclaved media after cooling at about 40 OC temperature.

Laminar air flow cabinets are used to create an aseptic working space blowing filter

sterilized air through an enclosed space. The air is first filtered through a coarse free

filter to remove large particles. It is then passed through HEPA filters which filters out

all particles larger than 0.3 lm. This sterilized air blows through the working area in a

cabinet at a constant speed of 1.8km/hr -1

. These filters not only eliminate dust and other

particles but also fungal and bacterial spores. Thus an aseptic environment is maintained

at the time of tissue inoculation.

4. Ultra violet sterilization: UV light sterilizes the interior portion of the inoculation

chamber and eliminates atmospheric contamination. Materials like nutrient media,

disposable plastic ware used for tissue culture and other similar materials are sterilized

using UV rays to remove the contaminates.5. Flame sterilization : Metalic instruments like foreceps, scalpels, needle, spatula are

sterilised by dipping in 95%. ethanal followed by flaming and cooling. This technique is

called flame sterilization. Autoclaving of metalic instrument is generally avoided as

they rust and become blunt. These instruments are repeatedly sterilized during their use

and time of inoculation to avoid contamination. The mouths of culture vessels are need

to be expose to flame prior to inoculation (or) sub culture

6. Chemical sterilization / Surface : The explant before its transfer to the nutrient

medium contain in the culture vessels is treated with an appropriate sterilizing agent to

inactivate the microbes present on their surfaces. This is known as surface sterilization.

The most commonly used sterilization for surface disinfection are


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Mercuric chloride 0.1% for 3-10min

Calcium hypochlorite 5% for 20 min

Sodium hypochlorite 0.5-1% for 15 min

Bromine water 1% for 2-10min

Chloramines 10-20% for 20-30min

Other H2O2 AgNo3 Antibiotic etc. are also used

The plant material to be sterilized is dipped in sterilant solution for prescribed period and

then the explant is taken out and washed with sterile distilled water for 2-3 times thoroughly

so as to remove all the traces of sterilant adhere to the plant material before its transfer to

nutrient media.

7. Wiping with 70% ethanol : The surfaces that can not be sterilized by other

techniques example plot form of laminar air flow cabinet, hands of operator etc are

sterilized by wiping them thoroughly with 70% alcohol and the alcohol is allowed todry.

Lecture No. 5*

Nutritional requirements of tissue culture

Preparation of composition of Murashige and Skoog medium

The isolated plant tissues are grown on a suitable artificially prepared nutrient medium

called culture medium. The medium is substrate for plant growth and it refers to the mixture

of certain chemical compounds of form a nutrient rich gel (or) liquid for growing cultures,

whether cells, organs (or) plantlets. The culture media has to supply all the essential mineral

ions required for in vitro growth and morphogenesis of plant tissue.

The major constituents of most plant tissue culture methods are :

1) Inorganic nutrients : micro and macro

2) Carbon source

3) Organic supplements

4) Growth regulators

5)

Solidifying agents

1) Inorganic Nutrients : A variety of mineral elements (salts) supply the macro & micro

nutrients required in the life of plant.

Elements required in concentration 0.5 ml / lit. are referred to as macro nutrients and

those required in less than 0.5 ml / lit. concentration are considered as micro nutrients


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Macronutrients

They include six major elements N.P.K.Ca Mg & S present as salts in the media which

are essential for plant cell and tissue growth

Nitrogen is the element which is required in greatest amount. It is most commonly

supplied as a mixture of Nitrate lons (KNO3)

Ammonium ions (NH4 NO3)

Phosphorous is usually supplied as phosphate ion of Ammonium, sodium and

Potassium salts. Other major elements Ca.Mg,S, are also required to be incorporated in the

medium

Micro nutrients

These are Mn.Zn. B, Cu. Mo. Fe. Co. I.

Iron is generally added as a chela te with with EDTA. (Ethylene Diamino Tetra Acetic

acid) In this form iron is gradually released and utilized by living cells and remains availableup to a PH of 8.


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Some of the elements are important for plant nutrition and their physiological function.

Element Function

Nitrogen (N2) Component of proteins nucleic acids some co-enzymes

Phosphrous (P) Component of nucleic acids energy transfer component of

intermediate in respiration and photosynthesis

Potassium (K) Regulates osmatic potential principal in organic cation

Calcium (Ca) Cell wall synthesis. Membrane function cell signally

Magnesium (Mg) Enzyme co-factor component of chlorophyl

Sulphur (S) Component of some amino acids (Methionine cysteine) some

co-factors

Chlorin (Cl) Required for photosynthesis

Iron (Fe) Electron transfer as a component of cytochromes

Managanese (Mn) Enzyme co-factorCobalt (Co) Component of some vitamins

Copper (Cu) Enzyme co-factor electron transfer reaction

Zinc (Zn) Enzyme co-factor chlorophyll biosynthesis

Molybdenum (Mo) Enzyme co-factor component of nitrate reductase.

Preparation of Nutrient Medium

The nutrients required for optimal growth of plant organ tissue and protoplast in vitro

generally vary from species to species eve n tissues from different parts of a plant may have

different requirements for satisfactory growth

Carbon source

Plants cells and tissues in culture medium lack autotrophic ability and therefore need

external carbon for energy. The most preferred carbon energy source in plant tissue culture is

sucrose. It is generally used at a conc of 2-5% while autoclaving the medium sucrose is

converted to Glucose and Fructose. In the process first Glucose is used and then Fructose,

Glucose supports good growth while fructose less efficient. Maltose Galactose then lactose

are mannose and the other sources of carbon. Most media contain myoenosital at a

concentration of approximately Ca 100 mg l-1 which improves cell growth.


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Organic supplements

Vitamins

Plants synthesize vitamins endogenously and these are used as catalysts in various

metabolic processes. When plant cells and tissues are grown in in vitro some essential

vitamins are synthesized but only in suboptimal quantities. Hence it is necessary to

supplement the medium with required vitamins and amino acids to get best growth of tissue.

The most commonly used vitamins is thiamine (vitamin B) the other vitamin which

improve growth of cultured plants are Nicotinic acid, Panthothenic acid Pyridoxin (B6) Folic

acid Amynobenzoic acid. (ABA)

Amino acids

Cultured tissues are normally capable of synthesing Amino acids necessary for various

metabolic processes. In spite of this the addition of Amino acid to the media is important for

stimulating cell growth in protoplast cultures and for establishing cell culture. Among theamino acids glycine is most commonly used Amino acids Glutamine Aspargine, Arginine

Cystine are the other common sources of organic Nitrogen used in culture media

Other organic supplements

These include organic extracts Eg:- Protein (casein) hydrolysate, coconut milk, yeast

& malt extract, ground banana, orange juice, Tomato juice, Activated charcoal. The addition

of activated charcoal to culture media stimulates growth and differentiation in orchids, Carrot

and Tomato Activated charcoal adsorbs inhibitory compounds & darkening of medium

occurs. It also helps in to reduce toxicity by removing toxic compounds

Eg:- Phenols produced during the culture permits un hindered cell growth

Antibiotics

Some plant cells have systematic infection of micro organisms. To prevent the growth

of these microbes it is essential to enrich the media with antibiotics

Eg:- Streptomycin or Kanamycin at low concentration effectively controls systemic

infection and do inhibit the growth of cell cultures

Growth regulators

These include auxins, cytokinins, gibberillins, ABA. The growth differentiation and

organogenesis of tissue occurs only on the addition of one (or) more of these hormones to the

medium.


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Auxins

Auxins have the property of cell division, cell elongation, elongation of stem,

internodes, tropism, Apical dominance abscission and rooting commonly used auxins are

IAA (Indole 3-Acetic Acid)

IBA (Indole 3-Butyric Acid)

2,4-D (Dichloro Phenoxy Acetic Acid)

NAA (Naphthylene Acitic Acid)

NOA (Naphthoxy Acitic Acid)

The 2,4-D is used for callus induction where as the other auxins are used for root

induction.

Cytokinins

Cytokinins are adenine derivaties which are mainly concerned with cell division

modification of apical dominance, and shoot differentiation in tissue culture. Cytokinins have been shown to activate RNA synthesis and to stimulate protein and the enzymatic activity in

certain tissues commonly used Cytokinins are

BAP (6-Benzylamino purine)

BA (Benzy adenine)

2ip (Isopentyl adenine)

Kinetine (6 – furfur aminopurine)

Zeatin (4 – hydroxy 3 methyl trans 2 butinyl aminopurine)

Gibberillins and Abscisic acid

GA3 is most common gibberillin used in tissue culture. It promotes the growth of the

cell culture at low density. Enhances callus growth and simulates the elongation of dwarf or

stunted plantlets formation from adventive embryos formed in culture.

ABA in culture medium either stimulates or inhibits culture growth depending on

species. It is most commonly used in plant tissue culture to promote distinct developmental

pathways such as somatic embryogenesis.

Solidfying agent

Gelling and solidifying agents are commonly used for preparing semisolid or solid

tissue culture media. Agar (polysaccharide obtained from marine sea weeds) is used to

solidify the medium. Normally 0.5-1% Agar is used in the medium to form a firm gel at the

pH typical of plant tissue culture media. Use of high concentration of agar makes the medium

hard and prevents the diffusion of nutrients into tissues.


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pH

Plant cells and tissues require optimum pH for growth and development in cultures.

The pH effects the uptake of ions, hence it must be adjusted below 5-6.0 by adding 0.1N

NaOH (or) HCLusually the pH higher than six results in a fairly hard medium where as pH

below five does not allow satisfactory solidification of medium.

Preparation of Nutrient Media

The nutritional requirement for optimum growth of plant organ, tissue and protoplast

in vitro generally vary from species to species even tissues from different parts of plant may

have different requirement for satisfactory growth. Therefore no single media as such can be

suggested as being entirely satisfactory for all types of in vitro culture. In order to formulate a

suitable medium for a new system a well known basal medium such as Ms (Murashigel and

skoog) B5 (gamborg et al) etc.

The composition of MS media is given below

Macro salts Concentration

NA4 NO3 ……………………………. 1.65 g

KNO3 ……………………………. 1.90 g

CaCl2 2 H2O ……………………………. 0.44 g

MgSO4 7H2O ……………………………. 0.37 g

KH2PO4 ……………………………. 0.17 g

Micro salts

FeSO4 7H2O ……………………………. 27.80 mg

Na2EDTA 2H 2O ……………………………. 33.60 mg

Kl ……………………………. 0.83 mg

K 3BO4 ……………………………. 6.20 mg

MnSO4 4H2O ……………………………. 22.30 mg

ZnSO4 7H2O ……………………………. 8.60 mg

Na2MoO4 2H2O ……………………………. 0.25 mg

CuSO4 5 H2O ……………………………. 0.025 mg

CoCl2 6H2O ……………………………. 0.025 mg

Organic supplements

Myoinositol ……………………………. 100.00 mg

Nicotinic acid ……………………………. 0.05 mg

Pyridoxine HCl ……………………………. 0.05 mg

Thiamine HCl ……………………………. 0.05 mg


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Glycine ……………………………. 0.20 mg

Sucrose ……………………………. 20.00 mg

Growth regulators As per need

Gelling agent (only for salid medium)

Agar ……………………………. (0.5-1%) 6-8 g/lig.

pH ……………………………. 5.8

By making minor quantitative and qualitative changes a new media can develop to

accommodate the specific requirements of the desired plant material

Preparation of the medium

The most suitable method for preparing media now-a-days is to use commercially

available dry powdered media. These media contains all the required nutrients. The powder is

dissolved in distilled water generally 10% less than final volume of medium and after addingsugar, agar and other desired supplements. The final volume is made up with distilled H 2o.

The pH is adjusted and media is autoclaved,

Another method of preparing media is to prepare concentrated stock solutions by

dissolving required quantities of chemicals of high purity in distilled water. Separate stock

solution are prepared for different media components

1. Major salts

2. Minor salts

3. Iron

4. Organic nutrients except sucrose

For each growth regulator a separate stock solutions is prepared. All the stock

solutions are stored in proper glass or plastic conta iners at low temperature in refrigerators.

Stock solution of Iron is stored in amber coloured bottles. Substances which are

unstable in frozen state must be freshly added to the final mixture of stock solution at the time

of medium preparation, Contaminated (or) precipitated stock solution should not be used.

The following sequential steps are followed for preparation of media

1)

Appropriate quantity of Agar and sucrose is dissolved in distilled water.

2) Required quantity of stock solution, heat stable growth harmones (or) other substances

are added by continuous stirring

3) Additional quantity of distilled water is added to make final volume of the medium.

4) While stirring the pH of the medium is adjusted by using 0.1 NaoH (or) HCL

5) If a gelling agent is used heat the solution until it is clear.


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6) medium is dispensed into the culture tubes, flasks, (or) any other containers.

7) The culture vessels are either plugged with non-absorbant cotton wool rapped in

cheese cloth or closed with plastic caps.

8) Culture vessels are sterilized in autoclave at 121oC 15Psi (1.06kg / cm

2)for about 15-

20 min

9) Heat labile constituents are added to the autoclaved medium after cooling to 30-40oC

under a Laminar airflow cabinet.

10) Culture medium is allowed to cool at room temperature and used or stored at 4oC

(1or2 days)

Lecture No. 6*

Types of media – Solid and liquid media – Advantages and limitations

Culture medium is a general term used for the liquid (or) solidified formulations upon

which plant cells, tissues (or) organs develop in the plant tissue culture. Thus normally the

explants are grown in two different types of media

1) Solid Medium

2) Liquid Medium

1) Solid Medium:-

A solidifying or a gelling agent is commonly used for preparing semisolid (or) solid

tissue culture medium. The plant material is placed on the surface of the medium. The tissue

remains intact and the cell multiplication is comparatively slow.

Advantages

1) solid medium is most widely used in plant tissue culture because of its simplicity

and easy handling nature. 2) Acquires sufficient aeration without a special device since the

plant material is placed on the surface of the medium.

Disadvantages

1) Only a part of the explant is incontact with the surface of the medium. Hence there

may be inequality in growth response of tissues and there may be a nutrient gradient between

callus and medium

2) There will be a gradiation in the gaseous exchange


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3) Solid medium represent a static system. Hence there will be polarity of the tissues

due to gravity and there will be variation in the availability of light to the tissues

4) Considerable damage to the tissues may occur during subculturing

5) Some physiological experiments which requires the immersion of tissues in the

culture medium can not be conducted by using the solid medium

2) Liquid medium:-

.All the disadvantage of solid medium can be overcome by use of liquid medium. It

does not contain a gelling or solidifying agent. So the plant material is immersed in the

medium either partially or completely. Liquid medium is used for suspension cultures and for

a wide range of research purposes.


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Advantages

1) The tissue is more easily supplied with nutrients.

2) The culture of plant tissue in an agitated liquid medium facilitates

a) Gaseous exchange

b) Removes any polarity of the tissue due to gravity

c) Eliminates nutrient gradient within the medium and at the surface of the

cells

3) Toxic waste products can be easily removed

4) Growth and Multiplication of cells tissues occur at a faster rate

5) There will be less damage to the tissues while sub-culturing

6) Isolation of secondary metabolites is easy

7) Liquid media are suitable for studies on the effect of any selective agent on

individuals cells.8) Therefore screening can be done at the cellular level for resistance to biotic and

abiotic stresses.

9) Liquid medium can be easily changed without re-culturing and are preferred for

some plant species whose explants exude phenols from their cut surfaces

Disadvantages

1. The explant gets submerged in liquid medium hence it requires some special devices

for proper aeration. Usually filter paper bridge may be used to keep the explant raised

above the level of the medium.

2. The cultures may be regularly aerated either by bubbling sterile air / gentle agitation

on a gyratory shaker

3. Needs to be sub-cultured frequently

Lecture No. 7*

Totipotency – Growth and differentiation in cultures –

Types of cultures – callus and suspension culturesExplant

A plant organ (or) an exised part used to initiate Tissue culture

Growth:-

An increase in size (vol/wt/length) due to cell division and subsequent enlargement

Differentiation:

The development of cells / tissues with the specific function and / or the regeneration

of organs / organ like structires / proembryos


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The phenomenon of mature cells reverting to a meristematic state and forming

undifferentiated callus tissue is termed as ‘De differentiation’

Callus

The ability of component cells of the callus to differentiate into a whole plant or a

plant organ is termed as Re-differentiation

Callus may be defined as an unorganized mass of loosely arranged parenchymatous

tissue which develop from parent cells due to proliferation of cells

Cellular Totipotency

The capacity of a plant cell to give rise to a whole plant is known as cellular

totipotency

Generally a callus phase is involved before the cells can undergo redifferentiation

leading to regeneration of a whole plant. The dedifferentiation cells can rarely give rise to

whole plant directly without an intermediate callus phase (Direct regeneration)Growth and differentiation although proceed together they are independent

Differentiation may be categorized into 2 groups

Structural 2) physiological

1) Structural differentiation

It is further distinguish into external and Internal differentia tion.

a) External

Most common example is root and shoot differentiation another familiar example of is

vegetative and reproductive phases of life cycle Further differentiation in reproductive organs

results in male and female organs


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b) Internal

This inc ludes differentiation of various types of cells and tissues. Differentied cells

mostly occur into groups forming different type of tissues

Eg:- Vascular tissues

c) Physiological

The variations in the structure between root and shoot are the expressions of

fundamental physiological differences

Cyto differentiation

In both plants and animals specialized cells perform different functions. This

specialization is known as cytodifferentiation.

The cells in a callus are parenchymatous in nature. The differentiation of these cells

into a variety of cells is required during re-differentiation of the cells into whole plants. This

re-differentiation of cells is known as cyto-differentiation. Eg:- Vascular tissue differentiation(Xylem and phloem)

Organogenic differentiation

For the regeneration of whole plant from cell (or) callus tissue cyto differentiation is

not enough and there should be differentiation leading to shoot bud and embryo formation.

This may occur either through organogenesis (or) somatic embryogenesis. Organogenesis

refers to the process by which the explants, tissues (or) cells can be induced to form root and

the (or) shoot and even whole plants. In other words formation of organs is called

organogenesis this may be categarised into 2 groups.

Rhizogenesis. The process of root formation

Caulogenesis. The process of shoot initiation

Somatic embroyogenesis

Development of embryos from somatic cells in culture whose structure is similar to

zygotic embryos found in seeds and with analogous embryonic organs such as cotyledons (or)

cotyledony leaves.

Factors affecting cyto-differentiation

1) Phytoharmones:- Auxins at low concentration stimulates xylogenesis. Cytokinins and

Gibberilins also stimulates tracheary element differentiation. When auxin and kinetin are used

together they have a synergistic effect


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2) Sugar:- Sucrose plays an important role in vascular tissue differentiation. Its concentration

directly effects the relative amounts of xylem and phloem formed in the callus in the presence

of low concentrations of auxin, 1% sucrose induces little xylem formation. Better xylem

differentiation with little (or) no phloem was observed when sucrose level was increased to

2% both xylem and phloem are differentiated at 2.5 to 3.5% sucrose concentration. With an

increase in sucrose concentration (4%) phloem was formed with little (or) no xylem.

3) Nitrogen:- Presence of Ammonia and Nitrate in the medium directly effects the

differentiation of tracheary elements

4) Physical factors :- The effect of light on vascular tissue differentiation varies between

cultured tissues. Temparature also effects cytodifferention. The temparature within 17-31oC

promotes vascular differention besides light and temparature other physical factors such as pH

of medium greatly effects cytodifferention

Callus growth patternGrowth of callus is measured in terms of increasc in fresh weight, dry weight (or) cell

nember A generalized growth pattern takes the form of a sigmoid curve

Three district phases can be observed during growth of the callus

Lag phase: a period of little (or) no cell division (Biomass remain unchanged)

Cell division followed by linear phase (logphase) :- a period of cell division and expansion

rate of division.

Stationary phase (or) regeneration phase:- As the nutrient supply of medium depleats, a

gradual cessation of cell division occurs. This phase is associated with the initiation of

structural organization of the cell which increases the production of secondary

metabolites.

Types of Cultures

1) Callus culture:-

callus culture may be derived from a wide variety of plant organs roots, shoots, leaves

(or) specific cell types. Eg:- Endosperm, pollen. Thus when any tissue (or) cell cultured on an

agar gel medium forms an unorganized growing and dividing mass of cells called callus

culture.

In culture, this proliferation can be maintained more (or) less indefinitely by sub-

culturing at every 4-6 weeks, in view of cell growth, nutrient depletion and medium drying.

Callus cultures are easy to maintain and most wide ly used in Biotechnology. Manipulation of

auxin to cytokinin ratio in medium can lead to development of shoots or somatic embryos

from which whole plants can be produced subsequently.


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Callus culture can be used to initiate cell suspensions which are used in a variety of

ways in plant transformation studies.

Callus cultures broadly speaking fall into one of the two categories.

1) compact 2) friable callus

In compact callus the cells are densly aggregated. Where as in friable callus the cells

are only loosly associated with each other and callus becomes soft and break a part easily. It

provides inoculum to form cell suspension culture.

Suspension culture

When friable callus is placed into a liquid medium (usually the same composition as

the solid medium used for callus culture) and then agitated single cells and / or small clumps

of few to many cells are produced in the medium is called suspension culture

Liquid cultures may be constantly agitated generally by a gyratory shaker of 100-250

rpm to facilitate aeration and dissociation of cell clumps into small pieces.Suspension cultures grow much faster than callus cultures, need to be sub-cultured at

every week, allow a more accurate determination of the nutritional requirement of cells and

even somatic embryos.

The suspension culture broadly grouped as 1) Batch culture 2) Continuous culture

1) Batch culture

A batch culture is a cell suspension culture grown in a fixed volume of nutrient culture

medium. Cell suspension increases in biomass by cell division and cell growth until a factor

in the culture environment (nutrient or oxygen availability) becomes limiting and the growth

ceases. The cells in culture exhibit the following five phases of a growth cycle.

i. Lag phase, where cells prepare to divide

ii. Exponential phase, where the rate of cell division is highest.

iii. Linear phase, where cell division shows but the rate of cells expansion increases.

iv. Deceleration phase, where the rates of cell division and elongation decreases.

v. Stationary phase, where the number and size of cells remain constant.

When cells are subcultured into fresh medium there is a lag phase. It is the initial

period of a batch culture when no cell division is apparent. It may also be used with reference

to the synthesis of a specific metabolite or the rate of a physiological activity. Then follows a

period of cell division (exponential phase). It is a finite period of time early in a batch culture

during which the rate of increase of biomass per unit of biomass concentration (specific

growth rate) is constant and measurable. Biomass is usually referred to in terms of the number

of cells per ml of culture. After 3 to 4 cell generations the growth declines. Finally, the cell


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population reaches a stationary phase during which cell dry weight declines. It is the terminal

phase of batch culture growth cycle where no net synthesis of biomass or increase in cell

number is apparent.

In batch culture, the same medium and all the cells produced are retained in the culture

vessel (Eg. culture flask 100-250 ml). the cell number or biomass of a batch culture exhibits a

typical sigmoidal curve. Batch cultures are maintained by sub-culturing and are used for

initiation of cull suspensions.

2) Continuous culture:-

These cultures are maintained in a steady state for a long period by draining out the

used (or) spent medium and adding the fresh medium. such subculture systems are either

closed (or) open type.

1) Closed:-

The cells separated from used medium taken out for replacement and added back tothe suspension culture. So that the cell biomass keeps on increasing

2) Open:-

Both cells and the used medium are takenout from open continuously cultures and

replaced by equal volume of fresh medium. The replacement volume is so adjusted that

cultures remain at sub-maximal growth indefinitely.

Lecture No. 8*

Micropropagation – Meristem culture – Procedure –Various approaches for shoot multiplication

In nature asexual reproduction takes place either by vegetative means (or) by

apomixis. The vegetative reproduction produces genetically identical plants and is widely

used for the propagation and multiplication of horticultural important plants. The

multiplication of genetically identical copies of a cultivar by asexual means is called clonal

propagation and a genetically uniform assembly of individual, derived originally from a

single individual by asexual propagation constitutes a clone . The word clone (Greek word

klone which means twig or slip like those broken off as propagules for multiplication) was

first used by Webber in 1903 to apply to cultivated plants. That were propagated vegetatively.

In vitro clonal propagation is called micro propagation

Techniques of Micro propagation:

The process of Micro propagation involves 4 distinct stages


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1. Selection of suitable explants, their sterilization and transfer to nutrient medium

for establishment / initiation of a sterile culture explant.

2. Proliferation of shoots from the explant on medium

3. Transfer of shoots to a rooting medium

4. Transfer of plants to soil / normal environment

1) Culture establishment / Initiation of a sterile culture explant

This stage consists of identification of mother plant and their preparation in such a

way that they provide more responsive explants suitable for establishment of contamination

free cultures. Cultures are initiated from various kind of explants such as meristem shoot tips,

nodal buds, internodal segments leaves, young inflorescene etc. but meristem, shoot tips and

nodal buds are most prefer for commercial micro propagation.

Meristems < 0.2mm (or) 0.2-0.4mm are devoid of pathogens and thus result in the

production of disease, virus free plants through micro propagation Selected explants aresurface sterilized and asptecally cultured on a suitable medium. During this stage a simple

medium without harmones and with low levels of both auxins and cytokinins is used.

In general the explants taken from Juvinile plants respond better. Cultures are

incubated in a room maintained at 25+ 1

oC temperature 16/8hr light/dark 3000-5000 lux light

intensity and 50-70% Relative humidity. After 2-4 weeks of incubation, the effective cultures

resume their growth

2) Proliferation of shoots from the explant on medium / Multiplication of propagules

Effective explants from stage I are subcultured on to a fresh medium. The time and

concentration of auxins and cytokinins in multiplication medium is an important factor

effecting the extent of multiplication. In vitro multiplication of shoots can be achieved by the

following main approaches

1. Multiplication through callus culture

2. Multiplication by adventitious shoots

3. Multiplication by apical and axillary shoots

4.

Multiplication by somatic embryo genesis

1) Multiplication through callus culture

1) A large number of plantlets can be obtained from callus either through shoot and root

formation (or) somatic embryogenesis. Mass production of callus followed by shoot

regeneration would be ideal method for large scale propagation of desired plants but

there are two serious drawbacks


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i) Due to repeated subculturing the capacity of mass calli to form the regenerable shoots is

diminished (or) even lost.

ii) Degree of aneuploidy, polyploidy and development of genetically aberrant cells

increased progressively resulting the regeneration of plant that differ from parent type.

Therefore multiplication of shoots through callusing is less preferred method. This approach

was successively used in citrus and oil palms.

2) Multiplication by adventitious shoots

Adventitious shoots are stem and leaf structures which develop naturally on plant at places

other than normal leaf axil regions. These structures include stems, buds, tubers, corms,

Rhizomes etc. In many horticultural crops, vegetative propagation through adventitious bud

formation is in commercial practice for Eg:- In nature shoots develop on of leaves of Begonia

and some other ornamental plants. In culture also similar type of adventitious shoot formation

can be induced by using appropriate combination of growth regulators in media. Adventitious buds can also be induced on the leaf and stem cuttings of even those species which are

normally not propagated vegetative Eg:- flax, Brassica species. Development of adventitious

shoots directly from excised organs is preferred for clonal propagation as compared to callus

formation method because in the former diploid individuals are formed but in the later often

cytologically abnormal plants are produced. However in varieties which are genetic chimeras

multiplication through adventitious bud formation may lead to splitting the chimeras to pure

type plants.


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Stages of micro propagation

Selection of an elite mother plant

Explant

STAGE-I

(Establishment)

Trimming

Surface sterilization and washing

maintenance of mother plant material

Establishment on appropriate growth medium

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Transfer to proliferation medium

STAGE-II(Proliferation)

Rapid shoot or embroyoid formation

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

STAGE-III Transfer to Rooting medium

(Rooting andHardening)

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

STAGE-IV Transfer of shoots or plant lets to(transfer to sterilized soil or artificial medium

normal environment)


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3) Multiplication by apical and axillary shoots

Apical shoots are those that occupy growing tip of shoots where as axillary shoots are

those that develop from their normal positions on the plant in a axil of each leaf. Every bud

has got potentiality to develop into a shoot. Apical dominance plays an important role in the

development of axillary buds and is governed by the growth regulators. In the species where

the apical dominance is very strong removal or injury of terminal bud is necessary for the

growth of axillary bud. To initiate culture, shoot tips are placed on the medium containing

low levels of cytokinins (0.05 to 0.5 mg/lit BAP) and auxin (0.01 to 0.1mg/lit, IBA), the level

of cytokinins is progressively raised at each subculture, until the desirable rate of proliferation

is achieved, In cultures, the rate of shoot multiplication can be enhanced by culturing the shot

tips on the medium containing cytokinins In such cases the cult ured explant transforms into

the mass of branches. By further subculturing, shoot multiplication cycle may be repeated and

culture may be maintained round the year and proliferating shoots of many species have beenmaintain upto 10-15 years.

The enhanced axillary branching method of shoot multiplication may be initially

slower than the other two methods. But each passage, the number of shoots increased. This

method is popular for clonal propagation of crop plants because the cells of shoot apex are

uniformly diploid and are least susceptible to genetic changes

4) Multiplication by somatic embryo genesis

Development of embryos from somatic cells in culture, whose structure is similar to

zygotic embryos found in seeds and with analogous embryonic organs such as cotyledonary

leaves or cotyledons.

Multiplication by somatic embryogenesis in nature is generally restricted to intra ovular

tissues

Eg:- Any cell of gametophytic or sporophytic tissue around the embryo sac, cells of

nucellus or Integument of members of the family Rutaceac can develop into embryos.

However in plant tissue cultures embryos can develop even from somatic cells like epidermis,

parenchymatous cells of petioles (or) secondary phloem etc.

Somatic embryos are formed by either of the two ways (Sharp et al. 1980)

1) Direct embryogenesis the embryos initiate directly from explant tissue in the absence of

callus proliferation

2) Indirect embryogenesis :- Cell proliferation that means callus is formed from explant

from which embryos are formed. Somatic embryo genesis encompasses various stages

from callus initiation to embryo development and maturation and subsequently plantlet


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formation. The media composition of each of these stages are different, somatic

embryos can be used to produced artifical seeds. Somatic embryogenesis as a means of

propagation is seldom use because

1) There is a high probability of occurrence of mutations

2) The method is usually rather difficult

3) The chances of loosing regenerating capacity becomes greater with repeated

subcultures

4) Induction of embryogenesis is often very difficult (or) impossible with many plant

species

5) A deep dormancy in seeds often occurs in the somatic embryos that may be

extremely difficult to break.

3) Transfer of shoot to a rooting medium : Shoots proliferated during stage II are

transferred to a rooting medium. In general rooting medium has low salt. All cytokininsinhibit rooting.

Eg:- half (or) even 1/4th salts of M.S medium and reduced sugar levels.

In most species 0.1 – mg/lit NAA. IBA is required for rooting. The availability of IBA

induces primary / secondary roots where as NAA induces root hairs Generally individual

shoots are approximately 2cm are transferred to rooting medium plantlets with 0.5 –1cm

roots are usually transplanted into pots since longer roots tend to get damaged during the

transfer.

4) Trans fer of plants to soil / normal environment

transfer of plantlets to soil is the most critical step in micropropagation. The plantlets

are maintained under highly protected conditions in in vitro i.e. high humidity, low irradiance,

low CO2 levels and high sugar content.

The ultimate success of micro propagation on commercial scale depends on the

capacity in the transfer of plants to the soil at low cost and high survival rates. The

heterotrophic mode of nutrition and poor physiological mechanisms. lack of cuticle on leaves

to control water loss, tender the micro propagation plants vulnerable to the transplantation,

plants are acclamatised in suitable compost mixture (or) soil in pots under controlled

conditions of light temperature and humidity. Inside the Green house the plants increase their

resistance to moisture stress and disease. The plantlets have to become autotrophic in

constrast to their heterotrophic state induced in micro propagation culture.


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Transfer of plantlets to soil is the most critical step in micro propagation. The plant

lets are maintained under highly protected conditions in in vitro i.e. high humidity, low

irradiance, low CO2 levels and high sugar content.

Shoot Meristem Culture

Cultivation of axillary or apical shoot meristems, particularly of shoot apical

meristem, is known as meristem culture. The shoot apical meristem is the portion lying distal

to the youngest leaf primordium, it is upto about 100 µm in diameter and 250 µm in length.

The first application of meristem culture was to obtain virus-free plants of dahlias; in 1952,

Morel and Martin isolated 100µm long meristem from virus -infected plants, and cultured

them to obtain virus-free shoots. Since then the technique of meristem culture has been

greatly refined and used for obtaining plants free from viruses, viroids, mycoplasma and even

fungi and bacteria in a range of crops. In India, some valuable clones of potato, sugarcane,

etc. have been freed from virus infections through meristem culture. Care must be taken toremove the apical meristem with as little surrounding tissue as possible to minimize the

chances of virus particles being present in the explant. This application of meristem culture is

of great value, particularly in the maintenance of breeding materials and germplasm

exchange, which are invaluable for any breeding programme.

Procedure of Meristem culture

1. Considerable expertise is required to dissect out the shoot apical meristem with

only one or two leaf primordia (100-500 µm in length).

2. Care has to be taken to prevent desiccation, and contamination by the virus present

in the surrounding tissue.

3. Generally, growth regulators (usually, small amounts of an auxin and a cytokinin)

are added to the medium to support shoot growth from the cultured meristems.

4. In general, the larger the meristem explant, the greater the chances of its survival

and shoot development. But the risk of infection by the virus also increases with

explant size

5.

Therefore, a compromise has to be reached between these two opposing forces in

deciding the explant size.

6. Viruses are eliminated by thermotherapy of whole plants, in which plants are

exposed to temperatures between 35-40oC for a few minutes to several weeks

depending on the host-virus combination.


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7. Thermotherapy is often combined profitably with meristem culture to obtain virus-

free plants. In general, shoot-tips are excised from heat-treated plants, but cultured

meristems may also be given thermotherapy; the former is preferable since larger

explants can be safely taken from heat-treated plants.

8. A prolonged exposure to a low temperature (5oC), followed by shoot-tip culture,

has proved quite successful in virus elimination; this is often called cryotherapy.

9. Some chemicals, e.g., virazole (ribavirin), cucloheximide, actinomycin D, etc.,

which interfere with virus multiplication, may be added into the culture medium

for making the shoot-tips free from Viruses; this is known as chemotherapy.

Applications and limitations

1. Virus elimination generally improves the yield by 20-90% over infected controls.

2. Virus-free plants serve as excellent experimental materials for evaluating the

detrimental effects of infections by various viruses.3. The virus free bulbs grew more rapidly, plants were more vigorous, and they

produced a greater number of larger flowers that had richer colour than the virus

infected stock.

4. The virus -free plants are deliberately infected by known viruses, and effects of the

infection on performance of the host are assayed.

5. Meristem culture can also help eliminate other pathogens, e.g., mycoplasmas,

bacteria and fungi. Bacteria and fungi present in explants show up when they are

cultured in vitro since tissue culture media provide excellent nutrition for the

microbes.

6. Meristem culture has been used to eliminate systemic bacteria form Diffenbachia

and Pelargonium, and Fusareum roseus from carnations.

Lecture No. 9

Micropropagation – Problems - Applications - Advantages and limitations

Problems

1) Microbial contaminationBacterial and fungal contamination in culture do not allow propagules to grow and

contaminated cultures have to be usually discarded. Such a problem can be overcome by

growing the donar plant in growth chamber, by effective sterilization of explants, by

performing inoculation in the laminar air flow cabinets and by using sterlised surgical


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instrument. Fumigation of inoculation with dilute formaldehyde solution helps to minimise

this problem.

2) Callusing

Callus formation is highly undersirable as it often effects the normal development of

shoots and roots and may lead to variability among the regenerated plants

Additon of tri-iodo-benzoic acid, flurogauicinol and flurorizin into the culture medium

(or) reduction of inorganic salt concentration helps in overcoming this problem

3) Tissue culture induced variation

The Micro propagation plants exhibit genetic (or) epigenetic variations which may be

a major problem in getting true to type plants. It can be controlled by careful selection of

initial explant, that is selecting meristems and controlling the cultural environment favouring

slow multiplication rates

4) Browning of mediumIn may species phenolic substances

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